MDMA Intoxication in a Potential Organ Donor with Cardiac Arrest.

Amphetamine and its derivatives' consumption is still an important public health issue, namely in terms of compounds variability and disposition to consumers. However, some of them, like 3,4-methylenedioxymethamphetamine (MDMA), still live in the illicit market, with continuous success. Nevertheless, there is always new information and data on MDMA intoxication, both in vivo and in postmortem context. The authors report an intoxication case with MDMA, in an 18-year-old male, considered a potential organ donor after a cardiac arrest. Whole blood samples were collected in vivo, at the emergency room (ER), and postmortem, at the National Institute of Legal Medicine and Forensic Sciences. After a general screening procedure, samples were extracted by solid phase extraction (OASIS® MCX), followed by gas chromatography-mass spectrometry analysis. The whole blood postmortem sample was positive for lidocaine (<500 ng/mL), compatible with the ER intervention, and positive for MDMA (2278 ng/mL) and methylenedioxyamphetamine (MDA) (49 ng/mL), while whole blood samples collected in vivo (during the maintenance of the individual under advanced life support), were positive for MDMA (504-1918 ng/mL) and MDA (20-89 ng/mL). Samples were negative for other substances, namely ethanol, other drugs of abuse and medicines. Results interpretation is pivotal to understand the behavior of the substance. Thus, in this case, MDMA postmortem behavior should be carefully evaluated, considering as possible influencers, in the specific context of the case, the time lapse between death verification, maintenance of the advanced life support and body manipulation for organ collection purposes. Also referred and discussed is the antemortem/postmortem ratio of MDMA obtained values, compared with literature references. There is no doubt that death was due to MDMA intoxication, but information from the analysis performed on the in vivo samples suggests that this type of sample should also be considered, in a complementary role, whenever possible.

Quantification of GHB and GHB-GLUC in an 1,4-butanediol intoxication: A case report.

Gamma-hydroxybutyric acid (GHB) is an endogenous compound with known action at the neural level. Its psychoactive effects led to an illicit use context including recreational purposes, muscle building effects in bodybuilders and drug-facilitated crimes, specifically in sexual assaults. Besides the misuse of the main compound, there are precursors like Gammabutyrolactone (GBL) and 1,4-butanediol (1,4-BD), usually non controlled substances, becoming a much easier way to obtain the target-compound. The authors present the first reported intoxication case in Portugal with 1,4-Butanediol, including the quantification of GHB and GHB-GLUC in serum, by GC-MS/MS TQD. A suspicious liquid and a serum sample were sent by an hospital ER and analysed by GC-MS-single quadrupole and GC-MS/MS TQD, respectively. A methodology including protein precipitation and GC-MS/MS TQD analysis was used to detect and quantify GHB and GHB-GLUC in serum. Toxicological analysis revealed the presence of 1,4-Butanediol in the liquid and GHB [171 mg/L] and GHB-GLUC [13,7 mg/L] in serum. The victim reverted the coma with no neurological sequelae. This was the first detected case, in Portugal, with 1,4-Butanediol, suggesting that it is important to be aware that consumers have different options to obtain illicit compounds, such as GHB. On the other hand, GHB-GLUC was identified and quantified for the first time in a real case, due to intoxication. This case highlights the importance of analysing all samples for active compounds, precursors and metabolites that can lead to the main intoxication origin.

A fast and reliable method for quantitation of THC and its 2 main metabolites in whole blood by GC-MS/MS (TQD).

An analytical method was developed for the simultaneous determination of Δ9- tetrahydrocannabinol (THC), 11-hydroxy-Δ9-tetrahydrocannabinol (11-OH-THC) and l1-nor-9-carboxy-Δ9-tetrahydrocannabinol (THC-COOH) in whole blood samples, for identification and quantitation purposes. Samples were prepared using solid-phase extraction, followed by gas chromatography/tandem mass spectrometry (GC-MS/MS) analysis in multiple reaction monitoring mode, with a total run time of 7.6min. MS/MS detection was achieved with two ion transitions per substance. The method was fully validated, including selectivity and capacity of identification, limit of detection and limit of quantitation, recovery, carryover, linearity (1-100ng/mL), intra-assay precision, inter-assay accuracy. The obtained results allowed its use in routine forensic analysis, with the application to real samples, both clinical and post-mortem.

A fast method for GHB-GLUC quantitation in whole blood by GC-MS/MS (TQD) for forensic purposes.

γ-Hydroxybutyric acid (GHB) is an endogenous compound with a historical use, both in licit and illicit terms. Importantly, the post-mortem behavior of GHB has been studied due to the possibility of using this compound as a biomarker for estimating the post-mortem interval (PMI). However, the post-mortem behavior of the recently discovered glucuronated GHB metabolite (GHB-GLUC) has not been studied. Nevertheless, GHB-GLUC may also have potential both to assist in PMI determination and also to increase the window of detection of GHB consumption. In this work, for the first time, a reliable method using GC-MS/MS for the quantification of GHB-GLUC in whole blood samples was developed and validated, with a simple, fast and cheap sample pretreatment. The method proved to be specific, precise, linear in a work range between 200 and 5000ng/mL, with LOD and LOQ of 52.65ng/mL and 200ng/mL, respectively, and an extraction recovery of 51%. Furthermore, the method was applied to a set of real post-mortem blood samples non-related with GHB intoxication and the obtained results were also discussed.

Antidepressants detection and quantification in whole blood samples by GC-MS/MS, for forensic purposes.

Depression is among the most prevalent psychiatric disorders of our society, leading to an increase in antidepressant drug consumption that needs to be accurately determined in whole blood samples in Forensic Toxicology Laboratories. For this purpose, this work presents a new gas chromatography tandem mass spectrometry (GC-MS/MS) method targeting the simultaneous and rapid determination of 14 common Antidepressants in whole blood: 13 Antidepressants (amitriptyline, citalopram, clomipramine, dothiepin, fluoxetine, imipramine, mianserin, mirtazapine, nortryptiline, paroxetine, sertraline, trimipramine and venlafaxine) and 1 Metabolite (N-desmethylclomipramine). Solid-phase extraction was used prior to chromatographic separation. Chromatographic and MS/MS parameters were selected to improve sensitivity, peak resolution and unequivocal identification of the eluted analyte. The detection was performed on a triple quadrupole tandem MS in selected ion monitoring (SIM) mode in tandem, using electronic impact ionization. Clomipramine-D3 and trimipramine-D3 were used as deutered internal standards. The validation parameters included linearity, limits of detection, lower limit of quantification, selectivity/specificity, extraction efficiency, carry-over, precision and robustness, and followed internationally accepted guidelines. Limits of quantification and detection were lower than therapeutic and sub-therapeutic concentration ranges. Overall, the method offered good selectivity, robustness and quick response (<16min) for typical concentration ranges, both for therapeutic and lethal levels.

Comparison of endogenous GHB concentrations in blood and hair in death cases with emphasis on the post mortem interval.

Gamma-hydroxybutyric acid (GHB) is an endogenous compound which has a story of clinical use and illicit abuse since the 1960's. The possibility to use a multi-sample approach for GHB evaluation, including whole blood and hair, to better characterize a forensic toxicology case and evaluate a possible causal association with the death is an exciting up-to-date issue. In addition, its post-mortem behaviour, namely regarding degradation and metabolism, has been increasingly investigated as a putative biomarker for post-mortem interval (PMI) estimation. Thus, in order to contribute to clarification of this specific aspect, whole blood and hair post-mortem GHB levels were evaluated in 32 real cases with previous information on death and autopsy data. The results obtained suggest that the PMI (until 5 days between death and sampling) influences GHB whole blood concentration, but not GHB levels in hair samples. No differences were encountered for the other parameters evaluated, including age, gender, cause of death and presence or absence of substances. This study brings new insights regarding the usefulness of GHB levels in forensic toxicology, which might be further strengthened with larger, but comparable, studies from other laboratories and institutions in the context of legal medicine.

A fast and reliable method for GHB quantitation in whole blood by GC-MS/MS (TQD) for forensic purposes.

Gamma-hydroxybutyric acid (GHB) is an endogenous compound with a story of clinical use since the 1960s. However, due to its secondary effects, it has become a controlled substance, entering the illicit market. A fully validated, sensitive and reproducible method for the quantification of GHB by methanolic precipitation and GC-MS/MS (TQD) in whole blood is presented. Using 100µL of whole blood, obtained results included a LOD and LLOQ of 0.1mg/L and a recovery of 86% in a working range between 0.1 and 100mg/L. This method is sensitive and specific to detect the presence of GHB in small amounts of whole blood (both ante-mortem or post-mortem), and is, to the authors' knowledge, the first GC-MS-MS TQD method that uses different precursor ions and product ions for the identification of GHB and GHB-D6 (internal standard). Hence, this method may be especially useful for the study of endogenous values in this biological sample.

Analysis of organophosphorus pesticides in whole blood by GC-MS-µECD with forensic purposes.

In the present work, two multi-residue methods for the determination of ten organophosphorus pesticides (OPs), namely chlorfenvinphos, chlorpyrifos, diazinon, dimethoate, fenthion, malathion, parathion, phosalone, pirimiphos-methyl and quinalphos, in post-mortem whole blood samples are presented. The adopted procedure uses GC-MS for screening and quantitation, and GC-µECD (electron capture detector) for compound confirmation. Three different Solid Phase Extraction (SPE) procedures for OPs with Oasis(®) hydrophilic lipophilic balanced (HLB) and Sep-Pak(®) C18 cartridges were tested, and followed by GC-µECD and GC-MS analysis. The Sep-Pak(®) C18 cartridges extraction procedure was selected since it generated analytical signals 5 times higher than those obtained with the two different Oasis(®) HLB cartridges extraction procedures. The method has shown to be selective for the isolation of selected OPs as well as to the chosen internal standard (ethion) in postmortem blood samples. Calibration curves between 50 and 5000 ng/mL were prepared using weighted linear regression models (1/x(2)). It was not possible to establish a working range for fenthion by GC-µECD due to the lower sensitivity of the detector to this compound, whereas for pirimiphos-methyl it was set between 500 and 5000 ng/mL. The limit of quantitation was established at 50 ng/mL for all analytes, except for pirimiphos-methyl by GC-µECD analysis (500 ng/mL). The average extraction efficiency ranged from 72 to 102%. The developed methods were considered robust and fit for the purpose, and had already been adopted in the laboratory routine analysis.

Gamma-hydroxybutyric acid endogenous production and post-mortem behaviour - the importance of different biological matrices, cut-off reference values, sample collection and storage conditions.

Gamma-Hydroxybutyric Acid (GHB) is an endogenous compound with a story of clinical use, since the 1960's. However, due to its secondary effects, it has become a controlled substance, entering the illicit market for recreational and "dance club scene" use, muscle enhancement purposes and drug-facilitated sexual assaults. Its endogenous context can bring some difficulties when interpreting, in a forensic context, the analytical values achieved in biological samples. This manuscript reviewed several crucial aspects related to GHB forensic toxicology evaluation, such as its post-mortem behaviour in biological samples; endogenous production values, whether in in vivo and in post-mortem samples; sampling and storage conditions (including stability tests); and cut-off reference values evaluation for different biological samples, such as whole blood, plasma, serum, urine, saliva, bile, vitreous humour and hair. This revision highlights the need of specific sampling care, storage conditions, and cut-off reference values interpretation in different biological samples, essential for proper practical application in forensic toxicology.

Simultaneous analysis of some club drugs in whole blood using solid phase extraction and gas chromatography-mass spectrometry.

The use of psychoactive substances to improve social relations and increase body energy, in Rave Culture, has raised many legal and health public concerns, both for illicit trade and consumption. Therefore, forensic toxicology plays an important role in this area, mainly linked to the detection and quantitation of these substances, both in vivo and in post-mortem samples. In fact, at the moment, forensic sciences have been under public authorities' scrutiny and critical look, due to the increasing attention of the media and public opinion, always applying for the use of scientific knowledge to help solving forensic cases. However, forensic toxicology results are only reliable to solve legal cases if all the analytical methodologies used are appropriately validated. In this work, a methodology for the extraction and analysis of 7-aminoflunitrazepam, buprenorphine, flunitrazepam, ketamine, methadone, phencyclidine (PCP) and d-propoxyphene was developed for whole blood samples, with solid phase extraction (SPE), using OASIS(®) MCX SPE columns, and gas chromatography coupled to mass spectrometry. The procedure presented here proved to be reliable, specific, selective and sensitive, with good LODs and LOQs and good precision.The adoption of a SPE procedure with an automatic SPE extraction device, allowed an increased level of automation in sample treatment, being contemporarily less time-consuming, increasing productiveness, and allowing good recovery and appropriate selectivity being, also, simple and reproducible. The simultaneous detection and quantitation of all compounds by the same extraction and detection methodology is crucial and has a great potential for forensic toxicology and clinical analysis.

Cellulolytic potential of a novel strain of Paenibacillus sp. isolated from the armored catfish Parotocinclus maculicauda gut

A cellulolytic bacterial strain, designated P118, isolated from the gut of the tropical fish Parotocinclus maculicauda was identified as belonging to the genus Paenibacillus based on phenotypic and chemotaxonomic characteristics and the 16S rRNA gene sequence. The novel strain was Gram-positive, spore-forming and rod-shaped. Catalase but not oxidase was produced. Carboxymethylcellulose was hydrolyzed but starch or gelatin was not. Acetoin production was negative whereas nitrate reduction and urease production were positive. Many carbohydrates served as carbon sources for growth. MK-7 was the predominant isoprenoid quinone. Anteiso-C15:0 (38.73 percent) and C16:0 (20.85 percent) were the dominant cellular fatty acids. Strain P118 was closely related to Paenibacillus amylolyticus NRRL NRS-290, P. pabuli HSCC 492, P. tundrae Ab10b, P. xylanexedens B22a, and P. tylopili MK2 with 98.3-98.8 percent 16S rRNA gene sequence similarity. The results presented here suggest that strain P118 represents a novel species of the genus Paenibacillus and it is a potential strain for further studies concerning its role in the production of industrially important products from cellulosic biomass.

Cellulolytic potential of a novel strain of Paenibacillus sp. isolated from the armored catfish Parotocinclus maculicauda gut.

A cellulolytic bacterial strain, designated P118, isolated from the gut of the tropical fish Parotocinclus maculicauda was identified as belonging to the genus Paenibacillus based on phenotypic and chemotaxonomic characteristics and the 16S rRNA gene sequence. The novel strain was Gram-positive, spore-forming and rod-shaped. Catalase but not oxidase was produced. Carboxymethylcellulose was hydrolyzed but starch or gelatin was not. Acetoin production was negative whereas nitrate reduction and urease production were positive. Many carbohydrates served as carbon sources for growth. MK-7 was the predominant isoprenoid quinone. Anteiso-C15:0 (38.73%) and C16:0 (20.85%) were the dominant cellular fatty acids. Strain P118 was closely related to Paenibacillus amylolyticus NRRL NRS-290, P. pabuli HSCC 492, P. tundrae Ab10b, P. xylanexedens B22a, and P. tylopili MK2 with 98.3-98.8% 16S rRNA gene sequence similarity. The results presented here suggest that strain P118 represents a novel species of the genus Paenibacillus and it is a potential strain for further studies concerning its role in the production of industrially important products from cellulosic biomass.

Novel sex-related characteristics of the longsnout seahorse Hippocampus reidi Ginsburg, 1933

This paper presents novel sex-related characteristics in Hippocampus reidi, expressed as a prominent and pigmented keel (males only), and by a row of dorsolateral spots mostly (97.4 percent) found in males. While the keel was found even in the smallest males sampled, the dorsolateral spots appear to be associated with body size. Ex situ observations revealed that during courtship males highlighted both characters by turning pale their background body colour. Understanding such colour and behavioural patterns is relevant to increase our knowledge about sexual selection in a morphologically and biologically unique group of fishes.

O presente trabalho descreve novos caracteres sexuais em Hippocampus reidi, expressos como uma quilha proeminente e pigmentada (machos apenas), e uma série de máculas dorsolaterais, encontradas principalmente (97,4 por cento) em machos. Enquanto a ocorrência da quilha foi observada mesmo nos menores machos, a presença de máculas dorsolaterais parece estar associada ao tamanho do corpo. Observações ex situ mostraram que os machos realçam ambos os caracteres durante o comportamento de corte, ao tornar pálida a cor de base do corpo. Destaca-se a relevância da compreensão de tais padrões de colorido para o avanço no conhecimento relativo à seleção sexual em um grupo de peixes de morfologia e biologia singulares.

Population characteristics, space use and habitat associations of the seahorse Hippocampus reidi (Teleostei: Syngnathidae)

This paper provides a case study of a threatened seahorse species, Hippocampus reidi, highlighting the importance of using ecological information to assist conservation and management initiatives. Underwater visual sighting data (50 x 2m transect) gathered along the NE, SE and S portions of the Brazilian coast revealed an unequal distribution across localities, perhaps related to harvesting pressure, and a mean density of 0.026 ind.m-2. Our findings suggest some restricted spatial use by H. reidi, which was consistent with its estimated home range, and with re-sighting of specimens. Reproduction was recorded year-round, however productive peaks may exist. Components of habitat structure mostly used as anchoring points were mangrove plants, macroalgae, cnidarians, seagrass, sponges, and bryozoans. Conservation recommendations include: further characterization and mapping of habitats; assessment of availability and condition of microhabitats in selected areas, and studies on dispersal routes during initial life stages

Neste artigo apresenta-se um estudo de caso de uma espécie ameaçada de cavalo-marinho, Hippocampus reidi, enfatizando a importância de utilizar informações ecológicas nas ações de conservação e manejo. Dados de censo visual (transectos de 50 x 2m) obtidos nas porções NE, SE e S da costa brasileira revelaram uma distribuição heterogênea entre as localidades, talvez relacionada à pressão de coleta, e uma densidade média de 0.026 ind.m-2. Os resultados sugerem limitação no uso do espaço por H. reidi (consistente com a área vital estimada e com a re-avistagem de exemplares) e que a reprodução ocorre o ano inteiro, embora com possíveis picos. Os componentes da estrutura do habitat mais freqüentemente utilizados como pontos de ancoragem pelos cavalos-marinhos foram vegetação de mangue, macroalgas, cnidários, capim marinho e briozoários. Recomendações conservacionistas incluem caracterização adicional e mapeamento de habitats; avaliação da disponibilidade e condição dos microhabitats em áreas selecionadas, e estudos sobre rotas de dispersão durante as fases iniciais de vida